1. Field of the Invention
The present invention relates to a fermentation process to have a high productivity with a novel mutant of Pichia, more specifically, for preparing erythritol under optimal fermentation conditions for maximum erythritol production by optimizing the environmental conditions of culture such as pH, temperature and by controlling osmotic pressure using a two stage fermentation, in which osmotic pressure is adjusted to a low level during growth phase and to a relatively high level during production phase by adding continuously glucose and NaCl or KCl.
2. Description of Prior Art
Erythritol, a four carbon sugar alcohol, is a naturally occurring substance and is widely distributed in nature. Like most of the other polyols, it is a metabolite or storage compound for seaweeds and mushrooms. Fruits like melons, grapes and pears also contain erythritol. As it is often produced by bacteria, fungi, and yeasts, erythritol also occurs frequently in fermented food systems like wines or beers, and processed vegetables such as soy sauce or the oriental miso bean paste.
Erythritol is a moderately sweet bulking agent with 60 to 70 percent of the sweetness of sucrose in a ten percent solution. Its high negative heat of solution provides the crystalline material with a strong cooling effect. As it has a taste which is very close to sucrose and with no bitter aftertaste, it is ideal to improve the taste of a combination with intense sweeteners like aspartame.
Being a small molecule, erythritol has strong colligative properties, i.e. a strong freezing point depression and boiling point elevation effect as well as a high osmotic pressure. In combination with its low hygroscopicity and viscosity in solution, it is very useful to reduce and control the water activity of foodstuffs.
Erythritol production from its natural sources such as fruits and vegetables is impractical due to the relative small amounts. Erythritol can be chemically produced by reduction of meso-tartarate, oxidation and reduction of 4,6-o-ethylidene-D-glucose, hydrolysis of dealdehyde starch, or addition of hydrogen. Since erythritol production by the chemical methods has been found to be expensive, it is worthwhile to explore an alternative method for the effective production of erythritol using microorganisms.
Erythritol can be produced by microbial methods with the osmophilic yeasts, especially species of the genus Torulopsis, such as T. magnoliae, T. veratilis, and T. candida; Endomzycopsis chodati; Hansenula supelliculsa; Pichia miso; Monilliella tomentosa var. pollinis; Trigonopsis variabilis; Trichosporonoides; Candida zeylanoides; and Aureobasidium. Some bacteria such as Leuconostoc oenos can also produce erythritol. Monilliella tomentosa var. pollinis produced erythritol on a medium containing 35.7% glucose with 45.6% yield. Erythritol production using this strain did not apply to industrial scale due to by-products such as glycerol and ribitol. Industrial production of erythritol has been performed by a mutant of Aureobasidium. The mutant was isolated and developed by cooperative study of Nikken Chemical and National Research Institute of Japan. The mutant produced erythritol with 47.6% yield on a medium containing 22.5% glucose and 2 g/L.h volumetric productivity.
It was found that the most polyols producing strains can grow on the conditions of high osmotic pressure such as the high concentration of sugars and salts. This fact suggests that polyols production has the relation to osmotic pressure. Reed et al. reported that glycerol productivity was improved by culturing a glycerol producing strain under the conditions of high osmotic pressure. However, erythritol production by controlling osmotic pressure has not been reported.
Therefore, in this invention, a wild strain of Pichia sp., an isolated strain from the air in 40% sucrose solution at Woosuk University, Chonbuk, Korea was selected to produce erythritol. The wild strain was mutated with NTG (N-methyl-N'-nitro-N-nitroguanidine) treatment. One of mutants has superior properties to the wild strain in erythritol yield from glucose, volumetric productivity, and sugar tolerance. By using the mutant of Pichia sp., the effect of osmotic pressure on erythritol production was investigated and two-stage fermentation, in which osmotic pressure was adjusted to a low level during growth phase and to a relatively high level during production phase, was performed by adding continuously glucose and NaCl or KCl.